1. Field of the Invention
The present invention relates to the discovery of a new intercellular adhesion molecule, designated ICAM-3 which is involved in the process through which populations of leukocytes recognize and adhere to cellular substrates. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at the sites of inflammation and sites of immune responses.
The present invention further relates to the use of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2, to inhibit intercellular adhesion of cells of granulocyte, lymphocyte, or macrophage lineage. The use of such molecules provides a method for the treatment of specific and non-specific inflammation.
The invention also relates to therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly with HIV-1, in an individual who is exposed to HIV or infected by HIV through the administration of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus.
The invention also relates to a therapeutic method for suppressing the migration of HIV-1 infected cells from the circulatory system using ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus.
The invention also relates to a therapeutic method for suppressing T-cell death and "syncytia" formation in an individual infected with HIV using ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus.
The present invention relates to the use of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2, in the treatment of asthma.
The present invention additionally relates to molecules capable of binding to ICAM-3 (hereinafter anti-ICAM-3). The binding of an anti-ICAM-3 molecule to ICAM-3 is intended to modulate the biological functions associated with ICAM-3. The binding molecules of the present invention can be an antibody, a peptide, or a carbohydrate which is capable of binding to ICAM-3. Such binding molecules are useful in modulating the biological functions of ICAM-3.
The present invention also relates to the use of a anti-ICAM-3, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2, to inhibit intercellular adhesion of cells of granulocyte, lymphocyte, or macrophage lineage. The use of such molecules provides a method for the treatment of specific and non-specific inflammation.
The invention also relates to therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly with HIV-1, in an individual who is exposed to HIV or infected with HIV through the administration of a anti-ICAM-3, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus.
The invention also relates to a therapeutic method for suppressing the migration of HIV-I infected cells from the circulatory system using an anti-ICAM-3 agent, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 agent. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus.
The present invention further relates to the use of an anti-ICAM-3 agent, alone or in combination with anti-ICAM-1, and/or anti-ICAM-2, in the treatment of asthma.
2. Description of the Related Art
A. Leukocyte Attachment and Functions
Leukocytes must be able to attach to cellular substrates in order to properly defend the host against foreign invaders such as bacteria or viruses, see Eisen, H. W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, Pa. (1980), pp. 290-295 and 381-418) for a review of these functions. Leukocytes must be able to attach to endothelial cells so that they can migrate from the circulatory system to sites of inflammation. Furthermore, they must attach to antigen-presenting cells so that a normal specific immune response can occur, and finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur.
Recently, leukocyte surface molecules involved in mediating the above attachment mediated functions were identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T-cells (Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment related functions described above (Springer, T. et al., Fed. Proc. 44:2660-2663 (1985)). The molecules identified by those antibodies were called Mac-1 and Lymphocyte Function-associated Antigen-1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T. A. et al. Immunol. Rev. 68:111-135 (1982)). These two molecules, plus a third molecule, p150,95 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion (Keizer, G. et at., Eur. J. Immunol. 15:1142-1147 (1985)).
The above-described leukocyte molecules were found to be members of a related family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper. Med. 158:1785-1803 (1983); Keizer, G. D. Hal., Eur. J. lmmunol. 15:1142-1147 (1985)), termed the "CD-18 family" of glycoproteins. This glycoprotein family is composed of heterodimers having one alpha chain and one beta chain. Although the alpha chain of each of the antigens differed from one another, the beta chain was found to be highly conserved (Sanchez-Madrid, F. et at., J. Exper. Med. 158:1785-1803 (1983)). The beta chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd whereas the alpha chains were found to vary from 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)). Although the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al., (J. Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983)).
A group of individuals has been identified who are unable to express normal amounts of any member of this adhesion protein family on their leukocyte cell surface (Anderson, D. C. et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C. et al., J. Infect. Dis. 152:668-689 (1985)). Such individuals are said to suffer from "leukocyte adherence deficiency disease" ("LAD") (Anderson, D. C., et at., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)). Characteristic features of LAD patients include necrotic soft tissue lesions, impaired pus formation and wound healing, as well as abnormalities of adhesion-dependent leukocyte functions in vitro, and susceptibility to chronic and recurring bacterial infections. Granulocytes from these LAD patients behave in the same defective manner in vitro as do their normal counterparts in the presence of anti-CD18 monoclonal antibody. That is, they are unable to perform adhesion related functions such as aggregation or attachment to endothelial cells. More importantly, however, is the observation that these patients are unable to mount a normal inflammatory response because of the inability of their granulocytes to attach to cellular substrates. Most remarkable is the observation that granulocytes from these LAD patients are unable to get to sites of inflammation such as skin infections due to their inability to attach to the endothelial cells in the blood vessels near the inflammation lesions. Such attachment is a necessary step for extravasation.
Lymphocytes from these patients displayed in vitro defects similar to normal counterparts whose CD-18 family o5 molecules had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to an inability of their cells to adhere to cellular substrates (Anderson, D. C. et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C. et al., J. Infect. Dis. 152:668-689 (1985)). These data show that immune reactions are mitigated when lymphocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the CD-18 family.
Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells). This adherence has been found to require cell-cell contact which involves specific receptor molecules present on the cell surface of the leukocytes. These receptors enable a leukocyte to adhere to other leukocytes, to endothelial cells, and other non-vascular cells. The cell surface receptor molecules, LFA-1, Mac-1 and p150,95, have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including defective antibody responses.
Additionally, since leukocyte adhesion is involved in the process through which foreign tissue is identified and rejected, an understanding of this process is of significant value in the fields of solid organ transplantation such as kidney, non-solid organ transplantation such as bone marrow, tissue grafting, allergy and oncology.
B. Infection with HIV
HIV infection is the cause of AIDS. Many variants of HIV have been described: the major two are HIV-1 and HIV-2. HIV-1 is prevalent in North America and Europe and HIV-2 is prevalent only in Africa. The viruses have similar structures and encode proteins having similar function. HIV infection is believed to occur via the binding of a viral protein (termed "gp120") to a receptor molecule (termed "CD4") present on the surface of T4 ("T helper") lymphocytes (Schnittman, S. M. et al., J. Immunol. 141:4181-4186 (1988), which reference is incorporated herein by reference). After binding this receptor, the virus enters the cell and replicates, and in the process, kills the T cell. The destruction of an individual's T4 population is thus a direct result of HIV infection.
The destruction of the T cells results in an impairment in the ability of the infected patient to combat opportunistic infections. Although individuals afflicted with AIDS often develop cancers, the relationship between these cancers and HIV infection is, in most cases, uncertain.
Although the mere replication of the HIV virus is lethal to infected cells, such replication is typically detected in only a small fraction of the T4 cells of an infected individual. Recent results suggest more viremia occurs than had been previously estimated, and the T-cell infection frequency can be as high as 1%.
Several lines of research have elucidated other mechanisms through which the HIV virus mediates the destruction of the T4 population.
Apart from HIV replication, HIV infected cells can be destroyed through the action of cytotoxic, killer cells. Killer cells are normally present in humans, and serve to monitor the host and destroy any foreign cells (such as in mismatched blood transfusions or organ transplants, etc.) which may be encountered. Upon infection with HIV, T4 cells display the gp120 molecule on their cell surfaces. Killer cells recognize such T4 cells as foreign (rather than native cells), and accordingly, mediate their destruction.
HIV infection can also lead to the destruction of non-infected healthy cells. Infected cells can secrete the gp120 protein into the blood system. The free gp120 molecules can then bind to the CD4 receptors of healthy, uninfected cells. Such binding causes the cells to take on the appearance of HIV infected cells. Cytotoxic, killer cells recognize the gp120 bound to the uninfected T4 cells, conclude that the cell is foreign, and mediate the destruction of the cell.
An additional mechanism, and one of special interest to the present invention, with which HIV can cause T4 cell death is through the formation of "syncytia." A "syncytium" is a multinucleated giant cell, formed from the fusion of as many as several hundred T4 cells. Infection with HIV causes the infected cell to gain the ability to fuse with other T4 cells, either HIV infected, or uninfected healthy cells. The syncytium cannot function and soon dies. Its death accomplishes the destruction of both HIV infected and HIV uninfected T4 cells. This process is of special interest to the present invention since it entails the direct cell-cell contact of T4 cells. The ability of HIV-infected cells to form syncytia indicates that such cells acquire a means for fusing with healthy cells.
HIV infection, and especially HIV-1 infection, appears to influence cell surface expression of the leukocyte integrins and cellular adherence reactions mediated by these heterodimers (Petit, A. J., et al., J. Clin. Invest. 79:188 (1987); Hildreth, J. E. K., et at., Science 244:1075 (1989); Valentin, A., et al., J. Immunology 144:934-937 (1990); Rossen, R. D., et al., Trans. Assoc. American Physicians 102:117-130 (1989), all of which references are incorporated herein by reference). Following infection with HIV-1, homotypic aggregation of U937 cells is increased, as is cell surface expression of CD18 and CD11b (Petit, A. J., et at., J. Clin. Invest. 79:188 (1987)). HIV-1 infected U937 cells adhere to IL-1 stimulated endothelium in greater frequency than uninfected U937 cells; this behavior can be suppressed by treating the infected cells with anti-CD18 or anti-CD 11a monoclonal antibodies or by treating endothelial substrates with anti-ICAM-1 antibodies (Rossen, R. D., et at., Trans. Assoc. American Physicians 102:117-130 (1989)). Monoclonal antibodies to CD18 or CD11a have also been found to be able to inhibit formation of syncytia involving phytohemagglutinin (PHA)-stimulated lymphoblastoid cells and constitutively infected, CD4-negative T cells (Hildreth, J. E. K., et al., Science 244:1075 (1989)). Treatment of only the virus infected cells with anti-CD18, or anti-CD11a monoclonal antibodies was found to have little effect on syncytium formation, suggesting that these antibodies principally protect uninfected target cells from infection (Hildreth, J. E. K., et at., Science 244:1075 (1989); Valentin, A., et at., J. Immunology 144:934-937 (1990)). Valentin et al. (Valentin, A., et al., J. Immunology 144:934-937 (1990)) have recently confirmed these observations by demonstrating that monoclonal antibodies specific for CD18 inhibit syncytia formed when continuous T cell lines are co-cultured with HIV-1 infected U937 cells.
Although the mechanism through which monoclonal antibodies specific for CD18 or CD11a protect susceptible cells from fusing with HIV infected cells remains unknown, and is not necessary to an appreciation of the present invention, studies with radiolabeled gp 120 suggest that heterodimers containing CD18 do not provide a binding site for the virus (Valentin, A., et al., J. Immunology 144:934-937 (1990)). Thus, HIV infection involves cell-cell interactions, and/or viral-cell interactions which mimic such cell-cell interactions. The cell-cell interactions may result in the transport of cell-free virus or the transport of virus infected cells across endothelial barriers. Viral-cell interactions which mimic the cell-cell interactions may facilitate or enable free virus to attach to and/or infect healthy cells.
The present invention thus derives, in part, from the observation that HIV infection, and particularly HIV-1 infection, results in increased expression of the CD11a/CD18 heterodimer and its binding ligand. This increased expression is significant in that it enhances the ability of HIV-infected T cells to adhere or aggregate with one another (i.e. to undergo "homotypic aggregation"). Since such homotypic aggregation is not observed to occur among quiescent normal leukocytes, this discovery indicates that the expression of the CD11/CD18 receptors and/or ligands, such as ICAM-1, is required for such aggregation. LFA-1 must bind to ICAM-1 in order for homotypic aggregation to occur. As disclosed herein, ICAM-3 is the only member of the ICAM family of molecules which is expressed at a high level on resting T-cells. Only anti-ICAM-3 antibodies are capable of blocking the adhesion of T-cells to LFA-1 unless the T-cells are "activated". Therefore anti-ICAM-3 antibodies can be used to suppress aggregation of T-cells.
Additionally, anti-ICAM-3 antibodies may be used to block the adhesion processes of infected T-cells which permits HIV-1 to be transmitted from an infected cell to a healthy cell of an individual, and also permits or facilitates infection of healthy cells with free virus.
C. Migration of HIV Infected Cells
The migration and dissemination of leukocytes is important in protecting an individual from the consequences of infection. These processes, however, are also responsible for the migration and dissemination of viral-infected leukocytes. Of particular concern is the migration and dissemination of leukocytes infected with HIV. The migration of such cells results in the formation of extravascular foci, and may cause tumors and other abnormalities.
Histologic examination of affected organs reveals focal extravascular mononuclear cell infiltrates. Attempts to identify virus-infected cells in such infiltrates in the central nervous system have revealed the presence of HIV-1 infected cells. These studies have shown that the HIV-1 virus resides primarily in monocytes and macrophages, and other cells of this lineage (R. T. Johnson, et al. FASEB J. 2:2970 (1988); M. H. Stoler et at., J. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al. Science 233:215 (1986)).
The mechanisms which stimulate formation of extravascular infiltrates of HIV-1-infected monocytoid cells have not previously been well defined. The mechanisms may involve either the: transport of cell-free virus or the transport of virus across endothelial barriers within the cytoplasm of infected mononuclear cells.
Since infection with HIV-1 stimulates cell surface expression of molecules which facilitate adherence of leukocytes to vascular endothelial cells and the translocation of leukocytes from the blood to extravascular tissue sites (C. W. Smith et al., J. Clin. Invest. 82:1746 (1988), herein incorporated by reference) it has been proposed to use antibodies which inhibit cellular migration to prevent the dissemination of HIV infected cells (WO 90/13316).
D. Asthma: Clinical Characteristics
Asthma is a heterogeneous family of diseases. It is characterized by a hyper-responsiveness of the tracheobronchi to stimuli (Kay, A. B., Allergy and Inflammation, Academic Press, New York (1987); which reference is incorporated herein by reference). Clinically, asthma is manifested by the extensive narrowing of the tracheobronchi, by thick tenacious secretions, by paroxysms of dyspnea, coughing, and wheezing. Although the relative contribution of each of these conditions is unknown, the net result is an increase in airway resistance, hyperinflation of the lungs and thorax and abnormal distribution of ventilation and pulmonary blood flow. The disease is manifested in episodic periods of acute symptoms interspersed between symptom-free periods. The acute episodes result in hypoxia, and can be fatal. Approximately 3% of the general world population suffers from asthma.
Two types of asthma have been described: allergic asthma and idiosyncratic asthma. Allergic asthma is usually associated with a heritable allergic disease, such as rhinitis, urticaria, eczema, etc. The condition is characterized by positive wheal-and-flare reactions to intradermal injections of airborne antigens (such as pollen, environmental or occupational pollutants, etc.), and increased serum levels of IgE. The development of allergic asthma appears to be causally related to the presence of IgE antibodies in many patients. Asthma patients who do not exhibit the above-described characteristics are considered to have idiosyncratic asthma.
Allergic asthma is believed to be dependent upon an IgE response controlled by T and B lymphocytes and activated by the interaction of airborne antigen with mast cell-bound pre-formed IgE molecules. The antigenic encounter must occur at concentrations sufficient to lead to IgE production for a prolonged period of time in order to sensitize an individual. Once sensitized, an asthma patient may exhibit symptoms in response to extremely low levels of antigen.
Asthma symptoms may be exacerbated by the presence and amount of the triggering antigen, environmental factors, occupational factors, physical exertion, and emotional stress.
Asthma may be treated with methylxanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins, and ephedrine), glucocorticoids (such as hydrocortisone), inhibitors of mast cell degranulation (i.e. chromones such as cromolyn sodium) and anticholinergics (such as atropine).
Asthma is believed to involve an influx of eosinophils ("eosinophilia") into the tissues of the lung (Frigas, E. et al., J. Allergy Clin. Immunol. 77:527-537 (1986), which reference is incorporated herein by reference).
Insight into the immunological basis of asthma has been gained from bronchoalveolar lavage studies (Godard, P. et al., J. Allergy Clin. lmmunol. 70:88 (1982)), and studies of respiratory smooth muscle tissue denuded of epithelium (Flavahan, N. A. et al., J. Appl. Physiol. 58:834 (1985); Barnes, P. J. et al., Br. J. Pharmacol. 86:685 (1985)). Although these studies have not led to the elucidation of the mechanism underlying the immunology of asthma, they have led to the development of a generally accepted hypothesis concerning the immunological etiology of the disease (see, Frigas, E. et al., J. Allergy Clin. Immunol. 77:527-537 (1986)).
The hallmarks of the pathology of asthma are a massive infiltration of the lung parenchyma by eosinophils and the destruction of mucociliary capacity. The "eosinophil hypothesis" suggests that eosinophils are attracted to the bronchus in order to neutralize harmful mediators released by the mast cells of the lung. According to the hypothesis eosinophils are attracted to the bronchi where they degranulate to release cytotoxic molecules. Upon degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D which enzymatically neutralize the harmful mediators of the mast cell. However, these molecules also promote the destruction of the mucociliary apparatus, thus preventing the clearing of the bronchial secretions, and contributing to the lung damage characteristic of asthma.
Since asthma involves the migration of cells, it has been proposed to use antibodies which inhibit this migration to mitigate the effects of allergens in a subject (WO 90/10453).